The formation of amyloid plaques and subsequent neuronal degeneration are characteristic features of Alzheimer's disease (AD). However it remains unclear how amyloid plaques are formed and how they induce neuronal degeneration. The exposure of vascular endothelium to fl- amyloid protein (Afl) has been reported to result in endothelial cell dysfunction due to generation of free radicals. Recent studies suggest that toxic effects of amyloid precursor protein (APP) may be due to its ability to induce oxidative stress via generation of oxygen radicals. To the best of our knowledge, however, direct experimental proof of this has not been obtained. Our proposed project for the first time will directly test the hypothesis that expression of APP induces oxidative stress in bovine brain endothelial cells. This will be possible through use of: (i) a newly developed technique to quantitate oxidative stress in different classes of membrane phospholipids in live cells, and (ii) bovine brain endothelial cells transfected with APP695 as well as cells cotransfected with the MnSOD gene. In Specific Aim #1, we will determine whether bovine brain endothelial cells expressing APP will be producing oxidative stress in greater amounts than the wild-type cells. We will also correlate the amount of oxidative stress in the cells with cytotoxicity of expressed APP. In specific Aim #2, we will establish whether expression of MnSOD in the bovine brain endothelial cells cotransfected with APP695 results in protection against oxidative stress and cytotoxicity of APP. Overall, our results will provide new insight into mechanisms of oxidative stress and cytotoxic effects of APP in relation to its important role in etiology of Alzheimer's disease.